AJP - Endo Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 294: E1109-E1118, 2008. First published April 15, 2008; doi:10.1152/ajpendo.00016.2008
0193-1849/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/6/E1109    most recent
00016.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ueno, M.
Right arrow Articles by Miyamori, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ueno, M.
Right arrow Articles by Miyamori, I.

Cardiac overexpression of hormone-sensitive lipase inhibits myocardial steatosis and fibrosis in streptozotocin diabetic mice

Masami Ueno,1 Jinya Suzuki,1 Yasuo Zenimaru,1 Sadao Takahashi,1 Tsutomu Koizumi,2 Sakon Noriki,3 Osamu Yamaguchi,4 Kinya Otsu,4 Wen-Jun Shen,5,6 Fredric B. Kraemer,5,6 and Isamu Miyamori1

1Third Department of Internal Medicine, 2Laboratory Animal Resources, and 3Department of Pathology, University of Fukui, Fukui; 4Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan; 5Veterans Affairs Palo Alto Health Care System, Palo Alto; and 6Division of Endocrinology, Stanford University, Stanford, California

Submitted 9 January 2008 ; accepted in final form 10 April 2008


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intracellular lipid accumulation (steatosis) and resultant lipotoxicity are key features of diabetic cardiomyopathy. Since cardiac hormone-sensitive lipase (HSL) is activated in diabetic mice, we sought to explore a pathophysiological function of cardiac HSL in the development of diabetic cardiomyopathy. Transgenic (Tg) mice with heart-specific HSL overexpression were generated, and cardiac histology, function, lipid profile, and gene expressions were analyzed after induction of diabetes by streptozotocin. Electron microscopy showed numerous lipid droplets in wild-type (Wt) hearts after 3 wk of diabetes, whereas Tg mice showed no lipid droplet accumulation. Cardiac content of acylglycerides was increased ~50% with diabetes in Wt mice, whereas this was blunted in Tg hearts. Cardiac lipid peroxide content was twofold lower in Tg hearts than in Wt hearts. The mRNA expressions for peroxisome proliferator-activated receptor-{alpha}, genes for triacylglycerol synthesis, and lipoprotein lipase were increased with diabetes in Wt hearts, whereas this induction was absent in Tg hearts. Expression of genes associated with lipoapoptosis was decreased, whereas antioxidant protein metallothioneins were increased in diabetic Tg hearts. Diabetic Wt hearts showed interstitial fibrosis and increased collagen content. However, Tg hearts displayed no overt fibrosis, concomitant with decreased expression of collagens, transforming growth factor-β, and matrix metalloproteinase 2. Notably, mortality during the experimental period was approximately twofold lower in diabetic Tg mice compared with Wt mice. In conclusion, since HSL overexpression inhibits cardiac steatosis and fibrosis by apparently hydrolyzing toxic lipid metabolites, cardiac HSL could be a therapeutic target for regulating diabetic cardiomyopathy.

lipotoxicity; lipolysis; animal model; transgenic mice

CARDIOVASCULAR DISEASES, including heart failure and coronary artery disease, are the leading cause of morbidity and mortality for both type 1 and type 2 diabetic patients (4). However, a number of diabetic patients appear to develop cardiac disease in the absence of significant coronary artery disease, designated diabetic cardiomyopathy (37). Diabetic cardiomyopathy is characterized by myocyte hypertrophy, interstitial fibrosis, and increased content of triacylglycerol (TAG) and cholesterol in cardiac muscle (2, 35). These histological abnormalities are associated with functional problems, diastolic dysfunction in the early stage, and contractile dysfunction in the late stage, eventually leading to congestive heart failure.

Based on experiments using diabetic animal models, a number of mechanisms have been proposed to explain the pathogenesis of diabetic cardiomyopathy. 1) A defect of insulin signaling, as seen in type 1 diabetes, causes cardiac dysfunction with decreased fatty acid (FA) oxidation and increased immature muscle fibers (3). 2) Hyperinsulinemia, as seen in type 2 diabetes, contributes to myocyte hypertrophy via increased growth signaling. 3) Activation of the renin-angiotensin-aldosterone system leads to interstitial fibrosis by activating transforming growth factor (TGF)-β (36). 4) Chronic high glucose leads to increases in advanced glycosylation end products, polyol and hexsosamine flux, and diacylglycerol (DAG) content, which stimulate collagen cross-linking, oxidative stress, impaired Ca2+ handling, and PKC, respectively (32). 5) Sympathetic dysfunction causes compromised relaxation and contraction. 6) Increased FA availability and oxidation cause a number of detrimental effects on the heart, namely, "lipotoxicity" (43).

Recent studies have been focused on the importance of lipotoxicity in the pathogenesis of diabetic cardiomyopathy. In diabetes, increased FA oxidation produces excessive reactive oxygen species (ROS), including lipid peroxide. Physiological levels of ROS can be scavenged by the action of superoxide dismutase, glutathione peroxidase, and metallothioneins (MTs) (5). However, in diabetes, excess FA oxidation overwhelms the scavenging capacity, leading to mitochondrial damage and endoplasmic reticulum stress. Increased ROS also activate TGF-β, leading to synthesis of collagens and other extracellular matrix (ECM) and, eventually, cardiac remodeling (32).

In diabetic Zucker rats, it appears that excessive FA flux causes myocardial TAG accumulation, and excess FA released from TAG droplets stimulates ceramide synthesis. Ceramide then increases inducible nitric oxide synthase (iNOS) expression via NF-{kappa}B, and the resultant lipid peroxide leads to myocyte apoptosis (50). In addition, there is an increase in cardiac DAG and PKC activation in streptozotocin (STZ)-induced diabetic mice (18). The PKCβ and PKC{delta} activated by DAG stimulate TGF-β, leading to ECM synthesis and fibrosis (7). Thus intracellular accumulation of acylglycerides appears to play an important role in the pathogenesis of diabetic cardiomyopathy.

Intracellular lipolysis in cardiomyocytes is catalyzed by two major cytosolic lipases: hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) (16, 51). The classic enzyme, HSL, is expressed in many organs and cells, including adipose tissues, skeletal and cardiac muscles, and pancreatic β-cells (17). HSL activity is regulated by hormones, such as catecholamines, which stimulate its activity via phosphorylation of the protein by cAMP-dependent protein kinase. Phosphorylated HSL translocates from the cytosol to the surface of lipid droplets at the onset of lipolysis. Insulin inhibits HSL activity via dephosphorylation of HSL by phosphodiesterase 3B (23). ATGL is expressed primarily in adipose tissues and at a low level in cardiac muscle, although ATGL null mice have been shown to develop steatotic cardiomyopathy (14). HSL hydrolyzes TAG, DAG, and cholesterol ester (CE), with the highest hydrolytic activity against DAG, as evidenced by the fact that HSL null mice accumulate DAG in many tissues, including heart (13). In contrast to HSL, ATGL specifically removes the first FA from TAG, generating FA and DAG. Thus HSL and ATGL are thought to catalyze intracellular lipolysis cooperatively in the tissues expressing these lipases.

In diabetes, impaired insulin action stimulates adipose lipolysis, and we previously reported that mRNA and protein expression of HSL were also increased in adipose tissues of STZ-diabetic rats (42). Since heart expresses HSL, cardiac HSL might be expected to be activated in diabetes; however, there are a limited number of studies investigating the function and regulation of cardiac HSL in diabetes. In a previous study we generated transgenic (Tg) mice with tetracycline-inducible HSL overexpression in heart and reported that Tg mice lacked fasting-induced TAG accumulation in cardiomyocytes (40). In the current study we have sought to clarify the pathophysiological functions of cardiac HSL in the development of diabetic cardiomyopathy by using Tg mice with constitutive overexpression of HSL in the heart. The results show that HSL activation is beneficial in preventing cardiac steatosis and fibrosis by decreasing toxic lipid metabolites, with a resultant decrease in overall mortality.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of heart-specific HSL-overexpressing mice. Rat HSL cDNA was inserted under the myosin heavy chain (MHC)-{alpha} promoter, kindly provided by Dr. J. Robbins (University of Cincinnati Medical Center, Cincinnati, Ohio) (39) (see GoFig. 2A). The transgene was injected into embryos derived from C57BL/6 x DBA2 F1 background in the Division of Laboratory Animal Resources at the University of Fukui. Out of 30 survivors, 6 founders were identified by PCR screening followed by Southern blot analysis. The founders were backcrossed to C57BL/6 mice for six to seven generations. All animals were fed a chow diet ad libitum and were maintained on a 12:12-h light-dark cycle. All procedures were performed in accordance with National Institute of Health guidelines for the care and use of animals and were approved by the guidelines for animal experiments of the University o Fukui.


Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 1. Cardiac hormone-sensitive lipase (HSL) expression and activity were analyzed 3 wk after the induction of diabetes in C57BL/6 mice. A: HSL mRNA expression was analyzed by quantitative RT-PCR (RT-qPCR) using mRNA from mouse ventricle. HSL expression was normalized to GAPDH, and relative expressions are presented against control mice (Cont). DM, diabetic mice. B: HSL protein expression was analyzed by Western blotting. The expression was normalized to GAPDH, and relative expressions are presented against control mice (top). At bottom are representative blots for HSL and GAPDH for a loading control. C: cardiac HSL activity assayed as neutral cholesteryl ester hydrolase (NCEH). *P < 0.05 vs. control mice. Values are means ± SE for 5–8 mice in each group.

 

Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 2. Generation and characterization of heart-specific HSL-overexpressing mice. A: the transgene consists of a mouse promoter of myosin heavy chain (MHC)-{alpha}, rat HSL cDNA, and a fragment containing human growth hormone polyA. B: Western blot analysis against rat HSL in various tissues from wild-type (Wt) mice and a Tg line, designated as MH-1 Tg mice. Tg mice show robust HSL expression in a heart-specific manner. C: HSL activity in various tissues from Wt and MH-1 Tg mice. NCEH activity was increased 8-fold in the hearts of MH-1 Tg mice compared with Wt heart. *P < 0.05 vs. Wt mice. Values are means ± SE for 3 mice in each genotype.

 
Induction of diabetes. Twelve- to sixteen-week-old mice were injected intraperitoneally with 150 mg/kg of STZ every 3–4 days until plasma glucose concentrations were higher than 350 mg/dl. Urine glucose and ketone bodies were also tested. Vehicle-treated mice served as nondiabetic controls. Three weeks after diabetes was documented, mice were killed at 10:00–11:00 AM and analyzed. Each experimental group consisted of equal numbers of male and female mice.

Blood chemistry. Blood was drawn by puncturing the left ventricle or descending aorta. Plasma concentrations of nonesterified FA (NEFA), TAG, total cholesterol (T-Ch), and insulin were measured using commercially available kits (Wako, Osaka, Japan). Plasma glucose concentration was measured using FreeStyle (Nipro, Osaka, Japan).

HSL activity. HSL activity was determined as neutral cholesteryl ester hydrolase (NCEH) activity using a cholesteryl [14C]oleate emulsion as described previously (22). Protein concentration was determined using the Bradford method.

Western blot analysis. Immunoblot analyses were performed as previously described using rabbit anti-HSL antibody or anti-ATGL antibody (Cell Signaling Technology, Beverly, MA) and analyzed with a FluorChem IS-8000 (Alpha Innotech, San Leandro, CA) (22).

Histological studies. Hearts were fixed in formalin (10% vol/vol), embedded, and sectioned. Tissue collagens were stained with picrosirius red as previously described with minor modification (19). Briefly, the sections were incubated with 0.1% sirius red (Direct Red 80; Sigma-Aldrich, St. Louis, MO) in saturated picric acid for 1 h, washed with 0.1 M acetic acid for 20 min, and rinsed with PBS. The slides were mounted, and color images were captured with a light microscope using a digital camera (Olympus AX-80). Morphometric analysis was performed by scanning the whole area of sections using Mac Scope software (version 2.56; Mitani, Fukui, Japan). Electron microscopy was performed as previously described (41).

Collagen assay. The Sircol collagen assay (Biocolor, Belfast, Ireland) was used to measure soluble forms of collagen following the manufacturer's protocol. The collagen content in each sample was normalized by total protein concentration.

Tissue lipid content. Mice were anesthetized and hearts excised after a perfusion with 3 ml of PBS from the left ventricle. Lipids were extracted to measure content of TAG, T-Ch, free cholesterol (F-Ch), and NEFA using assay kits (Wako). Cardiac content of lipid peroxide was measured following a protocol for thiobarbituric acid-reactive substances (TBARS) as described previously with minor modification (29) and are expressed as picomoles of TBARS per microgram of protein. Cardiac DAG content was analyzed using a method described previously (33). Briefly, the samples were incubated with DAG kinase and [{gamma}-32P]ATP. The lipids were then separated by thin-layer chromatography. The band corresponding to phosphatidic acid was visualized and scraped, and radioactivity was counted.

Quantitative RT-PCR. Total RNA was extracted from cardiac ventricle using Trizol reagent (Invitrogen, Carlsbad, CA) and reverse-transcribed using Quantitect reverse transcription kit (Qiagen, Hilden, Germany). The target genes were amplified and analyzed in triplicate using TaqMan probes and ABI Prism 7000 SDS software (Applied Biosystems, Foster City, CA). The expression values of target genes were normalized with that of GAPDH.

Statistical analysis. All values are means ± SE. Significance was determined using ANOVA followed by Fisher's protected least significant difference. Differences in mortality were determined using the {chi}2 statistic test. P < 0.05 was considered significant, and the differences between genotypes or conditions are indicated.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HSL expression and activity in the heart of diabetic mice. To clarify the regulation of HSL in diabetic heart, C57BL/6 mice were injected with STZ to induce diabetes. Three weeks later, mRNA and protein expression of cardiac HSL were increased approximately twofold in diabetic hearts compared with control hearts (Fig. 1, A and B). In parallel, HSL activity was elevated 80% in the hearts of diabetic mice (Fig. 1C). These data indicate that cardiac HSL expression and activity are increased in diabetic mice.

Characterization of heart-specific HSL-overexpressing mice. To explore the pathophysiological function of activated HSL in the development of diabetic cardiomyopathy, we planned to induce diabetes in the tetracycline-inducible, heart-specific HSL-overexpressing mice that we generated in a previous study (40). However, inducible HSL expression declined with increasing generations. Since constitutive activation of HSL did not appear to cause any detrimental effects on the fetus, we reconstructed a transgene that harbors rat HSL cDNA under a MHC-{alpha} promoter to create Tg mice with constitutive expression of HSL in the heart (Fig. 2A). Out of six founders, we established a Tg line, designated as MH-1, which showed a high level of HSL expression in a heart-specific manner (Fig. 2B, faint expression in lung is in pulmonary vessels). In parallel to protein expression, HSL activity in Tg heart was eight times higher than that in wild-type (Wt) heart (Fig. 2C). General appearance, growth and fertility of Tg mice were normal. The F6 or F7 heterozygous Tg mice and their Wt littermates were injected with STZ to induce diabetes, and 3 wk later, mice were killed for analysis.

Characterization of diabetic animals. Characterization and plasma parameters of control and diabetic Wt and Tg animals are summarized in Table 1. Plasma glucose concentration in STZ-treated mice was elevated over 500 mg/dl, insulin declined >80%, and body weight was decreased 24% in both genotypes. Interestingly, plasma TAG concentration was increased 73% with diabetes in Wt mice, whereas this elevation was not observed in Tg mice. To test the possibility that HSL Tg mice have increased cardiac VLDL metabolism in diabetes that might explain their reduced plasma TAG concentration, we investigated cardiac VLDL-TAG uptake by injecting 125I-labeled rabbit VLDL. The result showed that cardiac VLDL uptake was not altered with diabetes or HSL expression, whereas it was decreased twofold in liver in both genotypes (data not shown). Plasma concentrations of NEFA and T-Ch were not altered in either genotypes after 3 wk of diabetes; however, we documented an approximately twofold elevation of plasma NEFA concentration between 1 and 2 wk of the experimental period (data not shown). Notably, the mortality during the experimental period was lower in diabetic Tg mice (12.2%) than in Wt mice (19.3%), and it was particularly apparent in the female group (Tg 9.4% vs. Wt 25.3%), providing strong evidence that HSL overexpression might be beneficial in diabetes. To investigate cardiac function, echocardiography was performed 3 wk after diabetes was documented (28). Although heart rate was decreased with diabetes in both genotypes, basic systolic function seemed to be unaltered in both conditions and genotypes (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 1. Animal characterization, plasma parameters, and mortality during the experimental period

 
Effect of HSL overexpression on cardiac steatosis. To study the effect of HSL overexpression on diabetic cardiomyopathy, we performed histological examinations on the left ventricle. Low magnification electron microscopy showed numerous lipid droplets were accumulated around clusters of mitochondria in cardiomyocytes of diabetic Wt mice (Fig. 3, B and F); however, Tg hearts accumulated virtually no lipid droplets in either control or diabetic conditions (Fig. 3, C, D, G, and H). Light microscopy with oil red-O staining also showed similar results (data not shown). No other morphological changes were observed in the hearts of Tg mice.


Figure 3
View larger version (83K):
[in this window]
[in a new window]

  		Fig. 3. HSL overexpression inhibited cardiac steatosis in streptozotocin (STZ)-induced diabetic mice. Representative electron micrographs depict the histological appearance of left ventricles from Wt and Tg mice after 3 wk of diabetes (A–D, magnification x2,740; E–H, magnification x18,400). Diabetic Wt hearts (B and F) accumulated lipid droplets (arrowheads) around clusters of mitochondria, whereas no lipid droplets were observed in similar regions of the left ventricle from diabetic Tg mice (D and H). We used 5 animals per group and studied 6 positions per sample.

 
Compatible with the histological findings, cardiac TAG content was increased 45% with diabetes in Wt mice, whereas the elevation was not observed in Tg mice (Fig. 4A). Cardiac DAG content was 40% lower in Tg hearts than in Wt hearts under control conditions and tended to be lower in Tg hearts than in Wt hearts with diabetes (Fig. 4B). Cardiac content of free FA and F-Ch were not significantly altered with diabetes or HSL overexpression (Fig. 4, C and D), and only trace amounts of CE were found (data not shown). Importantly, cardiac content of lipid peroxides was twofold lower in Tg hearts than in Wt hearts in both control and diabetic conditions (Fig. 4E).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 4. Cardiac lipid content. Lipids were extracted from ventricles and quantified as described in EXPERIMENTAL PROCEDURES. A: triacylglycerol (TAG). B: diacylglycerol (DAG). C: nonesterified fatty acids (NEFA). D: free cholesterol (F-Ch). E: lipid peroxide. *P < 0.05 between genotypes or conditions as indicated. Values are means ± SE for 5 mice in each group.

 
Gene expressions. We then analyzed cardiac mRNA expressions of genes related to FA metabolism by quantitative RT-PCR (Fig. 5, A–L). The expression of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), a key regulator of FA oxidation and TAG synthesis, was increased greater than twofold with diabetes in Wt hearts, whereas this increase was completely absent in Tg hearts (Fig. 5A). The expression of genes involved in TAG synthesis, glycerol-3-phosphate acyltransferase, and phosphatidic acid phosphatases 2A and 2B displayed a tendency to be increased with diabetes in Wt hearts, whereas their expressions were significantly lower in Tg hearts compared with Wt hearts with diabetes (Figs. 5, B–D). The expression of PPAR{gamma} was also increased with diabetes in Wt hearts, although the increase was blunted in Tg hearts (Fig. 5H). Among the lipases expressed in cardiomyocytes, lipoprotein lipase (LPL) was increased 80% with diabetes in Wt hearts, although this increase was completely absent in Tg hearts (Fig. 5E). The expression of endogenous HSL and ATGL were increased with diabetes in both Wt and Tg hearts (Fig. 5, F and G). Interestingly, ATGL protein expression was twofold lower in Tg hearts than in Wt hearts under control conditions, whereas its expression was similar in Wt and Tg hearts with diabetes (data not shown). The expression of fatty acid transport protein (FATP)-4 and -6 was lower in Tg hearts compared with Wt hearts with diabetes, whereas fatty acid translocase (FAT)/CD36 was significantly increased with diabetes in Wt hearts (Fig. 5, I–K). Expression of nuclear receptor Nur77, which has been shown to regulate many genes for FA oxidation, including uncoupling protein-2 and -3 (UCP2 and UCP3) (26), was increased with diabetes in Wt hearts, in contrast, it was elevated in Tg hearts in both conditions (Fig. 5L).


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 5. Cardiac expression of genes related to TAG synthesis (A–D), lipolysis (E–G), and FA uptake/oxidation (H–L). The mRNA expressions were analyzed by RT-qPCR and normalized to GAPDH. The relative expressions are presented against control Wt mice. A: peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}). B: glycerol-3-phosphate acyltransferase (GPAT). C: phosphatidic acid phosphatase 2A (PAP2A). D: PAP2B. E: lipoprotein lipase (LPL). F: endogenous HSL. G: adipose triglyceride lipase (ATGL). H: PPAR{gamma}. I: fatty acid transport protein-4 (FATP4). J: FATP6. K: fatty acid translocase (FAT)/CD36. L: nuclear receptor Nur77. *P < 0.05 between genotypes or conditions as indicated. Values are means ± SE for 5 mice in each group.

 
We next analyzed the expression of genes involved in lipoapoptosis and glucose-induced fibrosis. Among the genes related to lipoapoptosis, the expression of the key molecules NF-{kappa}B and iNOS were significantly lower in diabetic Tg hearts compared with Wt hearts (Fig. 6, A and B). In contrast, expression of the ROS scavengers MT-1 and MT-2 were markedly increased in diabetic Tg hearts compared with Wt hearts. It is known that high-glucose stimulates TGF-β function and matrix metalloproteinase (MMP)-2 cleaves latent TGF-β to release the active form of the protein. As shown in Fig. 6, E and F, expression of both TGF-β and MMP-2 was decreased 40–50% in diabetic Tg hearts compared with Wt hearts. In parallel, expression of type I, III, and XV collagens were all decreased 70–80% in Tg hearts compared with Wt hearts with diabetes (Fig. 6, G–I).


Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 6. Cardiac expression of genes related to apoptosis (A and B), oxidative stress (C and D), and fibrosis (E–I). The mRNA expressions were analyzed by RT-qPCR and normalized to GAPDH. The relative expressions are presented against control Wt mice. A: NF-{kappa}B. B: inducible nitric oxide synthase (iNOS). C: metallothionein-1 (MT1). D: MT2. E: transforming growth factor-β (TGF-β). F: matrix metalloproteinase-2 (MMP2). G: type I collagen {alpha}-1. H: type III collagen {alpha}-1. I: type XV collagen {alpha}-1. *P < 0.05 between genotypes or conditions as indicated. Values are means ± SE for 5 mice in each group.

 
To identify potential adverse effects of HSL overexpression, we studied cardiac mRNA expression of TNF-{alpha}, interleukin-6, atrial and brain natriuretic peptides, and MHC-β/{alpha}. However, expression of none of these genes was significantly altered in Tg mice compared with Wt mice. In addition, we studied potential macrophage infiltration in cardiac muscle, analyzing scavenger receptor type A1 and A2. Again, the expression was similar between Tg and Wt mice (data not shown).

Cardiac fibrosis. Since mRNA expression of collagens was markedly decreased in diabetic Tg hearts, we investigated cardiac fibrosis by histological examination with collagen staining. As shown in Fig. 7, A–D, interstitial collagens were significantly increased in diabetic Wt hearts. In contrast, this interstitial fibrosis was absent in diabetic Tg hearts. A morphometric analysis showed that the interstitial collagen mass, which includes cross-linked collagens, was increased 2.3-fold with diabetes in Wt mice. In contrast, the increase was totally absent in Tg heart (Fig. 7E). In addition, cardiac content of soluble collagens, which represents recently synthesized collagen pool, was increased ~30% in diabetic Wt mice, whereas this elevation was not observed in diabetic Tg mice (Fig. 7F). To test the effect of HSL overexpression on collagen degradation pathways, we analyzed mRNA expressions of MMP-9 and tissue inhibitor of metalloproteinase-1 and -2. However, these genes were not affected in any groups. In addition, the expression of aldose reductase, which is a key regulator in the polyol pathway, was not altered (data not shown).


Figure 7
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 7. Effect of HSL overexpression on cardiac fibrosis in STZ-diabetic mice (A–F) and a working hypothesis as to how HSL overexpression inhibits cardiac fibrosis (G). A–D: representative photomicrographs depicting interstitial fibrosis, stained in red, in cardiac ventricles. Tissue sections from Wt and Tg mice were stained with picro-sirius red. A: control Wt. B: diabetic Wt. C: control Tg. D: diabetic Tg. Magnification, x400. E: the amount of total collagen relative to control Wt mice was determined from morphometric analysis [n = 12 (4 mice x 3 sections in each group)]. F: cardiac content of soluble collagens. Values are means ± SE; n = 7–8 mice. *P < 0.05 between genotypes or conditions as indicated. G: HSL overexpression decreases cardiac content of lipotoxic acylglycerides and lipid peroxide. As a result, expression of fibrogenic molecules including TGF-β, MMP2, and collagens are decreased, maintaining a normal level of interstitial collagens even in the setting of high glucose. Induction of MTs assists this inhibitory pathway by scavenging reactive oxygen species (ROS).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the current study we have shown that cardiac overexpression of HSL is able to prevent histological, biochemical, and molecular changes associated with diabetic cardiomyopathy. It appears that an increase (~2-fold) in cardiac HSL expression and activity in diabetic heart (Fig. 1) is a self-adjusting mechanism in response to an energy shift from glucose to FA. In preliminary experiments we have documented that the maximum increase in cardiac HSL activity under physiological conditions was up to threefold when mice were refed after a 24-h fast. In contrast, the HSL activity in our Tg mice seems to be superphysiological (8-fold increased), which could have overcome diabetes-induced TAG accumulation, resulting in beneficial consequences as related to cardiomyopathy.

Several laboratories, including ours, have reported the phenotypes of tissue-specific HSL-overexpressing mice. In macrophage-specific HSL-overexpressing mice, increased NCEH activity in macrophages led to an increase in atherosclerotic lesions with a paradoxical increase in cellular CE content (10). In Tg mice with β-cell-specific HSL overexpression, reduction of ATP and increased UCP2 led to impaired insulin secretion (47). Interestingly, adipose HSL-overexpressing mice showed impaired catecholamine-induced lipolysis (25). Thus FA toxicity might have exerted adverse effects in each of these HSL Tg mouse models. On the basis of these reports, we expected to observe some evidence of cardiac lipotoxicity in the current Tg mice. However, none of the genes related to lipotoxicity was significantly altered in Tg hearts. Instead, cardioprotective genes MTs were markedly increased. These data provide evidence that cardiac HSL overexpression is beneficial rather than detrimental in diabetes.

Interestingly, cardiac HSL overexpression caused systemic effects in mortality and plasma TAG concentration. The mortality during STZ treatment was significantly reduced in female Tg mice. Since female mice are STZ resistant, they required more STZ injections than male mice. The average number of the STZ injections to induce diabetes was 1.6 ± 0.13 for male mice and 3.4 ± 0.15 for female mice. When the present data were analyzed in male and female groups separately, the female group tended to show more significant differences between genotypes compared with the male group. Although it is difficult to clarify the precise mechanism for this difference, we speculate that increased STZ injections might have exerted some cardiotoxicity on the top of the lipo- and glucotoxicity in female mice and that HSL overexpression ameliorated overall toxicity by decreasing ROS and increasing MTs. The cardioprotective function of estrogen also might have affected the female mortality (27).

Plasma TAG concentration was elevated with diabetes in Wt mice, whereas it remained normal in Tg mice. We first hypothesized that Tg hearts might have increased VLDL-TAG uptake with diabetes; however, the results of tracer experiments excluded this possibility. Other possible explanations might involve increased function of systemic or cardiac LPL and decreased hepatic VLDL production/secretion. Since interorgan cross talk has been reported (24, 44), cardiac HSL overexpression might have affected function of the tissues or molecules involved in plasma TAG metabolism. These possibilities remain to be investigated.

It has been reported that HSL-deficiency is cardioprotective. Park et al. (30) have shown that HSL knockout (KO) mice on a high-fat diet have increased cardiac glucose uptake and decreased TAG content compared with Wt mice (30). We interpret that these finding are due to impaired adipose lipolysis and resultant lower plasma FA concentrations in HSL KO mice. Indeed, we have documented 60% lower plasma FA concentrations and cardiac TAG content in the heart of fasted HSL KO mice (39a). Thus HSL KO mice appear to exhibit phenotypes related to impaired adipose lipolysis, masking the effects in other tissues expressing HSL. In contrast, since our animal model has HSL overexpression exclusively in the heart, we were able to identify the heart-specific effects of HSL overexpression in the setting of diabetes.

PPAR{alpha} is a ligand gated nuclear transcription factor that directly or indirectly regulates the expression of many proteins and enzymes modulating FA uptake and oxidation. The transcriptional regulation of PPAR{alpha} is not completely understood; however, it has been reported that PPAR{alpha} mRNA expression is increased by FA in the liver of STZ-diabetic rats (1) and by retinol in cultured adipocytes (45). Although PPAR{alpha} is a key regulator of FA metabolism in the heart, recent studies have shown that excess PPAR{alpha} (and PPAR{gamma}) function can have adverse effects on cardiomyocytes. Cardiac overexpression of PPAR{alpha} caused lipotoxic cardiomyopathy (11), whereas PPAR{alpha} KO mice were resistant to diabetic cardiomyopathy (12). Recently, it was reported that cardiac overexpression of PPAR{gamma} also leads to steatotic cardiomyopathy (38). A PPAR{alpha} agonist led to cardiomyocyte necrosis due to increased FA oxidation and oxidative stress (34) and to stimulated cardiac fibrosis after ischemia-reperfusion (8). In the current study, heart-specific HSL Tg mice did not show a diabetes-induced increase in mRNA of PPAR{alpha} and TAG synthetic genes, some of which are known to be PPAR{alpha} regulated (Fig. 5A). Thus blocking the upregulation of PPAR{alpha} (and PPAR{gamma}) is likely to be one of the mechanisms how HSL overexpression inhibits cardiac steatosis. In addition, since HSL hydrolyzes retinyl ester, and given that its product, retinol, which is a ligand of retinoid X receptors, plays an important role in the development and function of cardiomyocytes (31), accelerated retinyl ester metabolism might be also involved in the altered expression and function of PPAR{alpha} in HSL Tg heart.

Expression of LPL, which is also known to be a PPAR{alpha} target gene, and FATPs was also altered in parallel to PPAR{alpha}. Cardiac LPL hydrolyzes TAG in circulating lipoproteins, and FATPs facilitate FA uptake into cardiomyocytes, playing a role in TAG accumulation in cardiomyocytes. Given the function of these molecules and the reports showing that overexpression of cardiac LPL or FATP1 can cause steatotic cardiomyopathy (6, 49), suppression of these genes might also contribute to the inhibition of steatosis in HSL Tg mice. The question then is, how does HSL overexpression inhibit PPAR{alpha} upregulation in diabetes despite an expected increased FA availability mediated by the action of HSL? One possibility would be that in HSL Tg heart, FA oxidation is activated so that released FA are immediately oxidized before they stimulate PPAR{alpha} expression. Supporting this hypothesis, mRNA expression of Nur77 was significantly increased (Fig. 5L) and that of UCP2 was 60% increased in HSL Tg hearts (significant only in the female group; data not shown). Another possibility is that FA released by HSL action might be rapidly diffused out of the cells via a transmembrane flip-flop mechanism (15). The modest increase in cardiac FA content in Tg mice (Fig. 4C) might support the participation of this process. The metabolic fates of FA released by HSL should be investigated.

MTs are potent antioxidant proteins that scavenge free radicals and toxic metal ions in many tissues, including heart (5). Overexpression of MT2 has been shown to protect cardiomyocytes from apoptosis by inhibiting caspase-3 activation in STZ-diabetic mice (48) and to rescue doxorubicin or high fat-induced cardiomyopathy by scavenging ROS (9, 21). In the current study, mRNA of both MT1 and MT2 were markedly increased in diabetic Tg hearts (Fig. 6, C and D). Thus the upregulation of MTs might have contributed to the inhibition of cardiac fibrosis and protection against oxidative stress.

TGF-β is known to be a key regulator inducing cardiac fibrosis in diabetes, hypertension, and ischemic heart disease (7, 36). It has been reported that TGF-β expression is increased in the hearts of diabetic mice (46). In the diabetic HSL Tg mice, expression of MMP2 and TGF-β were decreased, and this was paralleled by a marked decrease in collagen expression and content (Figs. 6 and 7). These findings are quite similar to those in STZ-diabetic mice treated with an angiotensin receptor blocker (46). In our HSL Tg mice, a reduction in lipid peroxide content due to increased actions of HSL and MTs might have suppressed the MMP2/TGF-β pathway.

TGF-β can be regulated by several other mechanisms. Diabetic mice have been shown to have cardiac DAG accumulation and evidence of PKCβ activation, which stimulates the TGF-β pathway (18). Given these findings, we studied membrane-bound DAG-sensitive PKCs by performing Western blotting. However, the DAG-sensitive PKC{alpha}, PKCβ, and PKC{epsilon} were not altered between Wt and Tg hearts (data not shown). Although a longer period of diabetes might have shown greater DAG accumulation and significant activation of PKCs (20), the results suggest that lipid peroxides are likely to have a greater role than PKCs in stimulating TGF-β-induced fibrosis in this animal model.

Figure 7G summarizes a working hypothesis how HSL overexpression inhibits cardiac fibrosis in diabetic animals. HSL overexpression hydrolyzes intracellular acylglycerides, leading to a decrease in toxic lipid peroxides. Increased MTs also support scavenging ROS/lipid peroxide. As a result, MMP2 and TGF-β are decreased, leading to inhibition of collagen expression, synthesis, and finally, fibrosis. It has been reported that the PPAR{gamma} agonist rosiglitazone improves diabetic cardiomyopathy by reducing cardiac TAG content in Zucker diabetic rats (50). In that report, rosiglitazone also improved plasma glucose and FA concentrations. In the current study, our animal model shows that decreasing intracellular acylglycerides has a protective effect against fibrosis, even in the presence of a high glucose concentration. Although lipotoxicity is only one possible mechanism, and others might exist, the current study supports the importance of controlling lipotoxicity for preventing the development of diabetic cardiomyopathy. Since HSL has been shown to exert beneficial effects without FA toxicity in the heart, there is a potential for cardiac HSL to be a clinical target to control or prevent diabetic cardiomyopathy.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by a research grant from Japanese Ministry of Education, Culture, Sports, Science and Technology (to J. Suzuki).


   ACKNOWLEDGMENTS
 
We thank Dr. J. Robbins for providing a clone containing MHC-{alpha} promoter. We offer special thanks to the skillful technical assistance of N. Yamaguchi, M. Uno, and H. Maeda.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Suzuki, Third Dept. of Internal Medicine, Univ. of Fukui, Faculty of Medical Science, Fukui 910-1193, Japan (e-mail: jinya{at}u-fukui.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Asayama K, Sandhir R, Sheikh FG, Hayashibe H, Nakane T, Singh I. Increased peroxisomal fatty acid beta-oxidation and enhanced expression of peroxisome proliferator-activated receptor-alpha in diabetic rat liver. Mol Cell Biochem 194: 227–234, 1999.[CrossRef][Web of Science][Medline]
  2. Asbun J, Villarreal FJ. The pathogenesis of myocardial fibrosis in the setting of diabetic cardiomyopathy. J Am Coll Cardiol 47: 693–700, 2006.[Abstract/Free Full Text]
  3. Belke DD, Betuing S, Tuttle MJ, Graveleau C, Young ME, Pham M, Zhang D, Cooksey RC, McClain DA, Litwin SE, Taegtmeyer H, Severson D, Kahn CR, Abel ED. Insulin signaling coordinately regulates cardiac size, metabolism, and contractile protein isoform expression. J Clin Invest 109: 629–639, 2002.[CrossRef][Web of Science][Medline]
  4. Bell DS. Diabetic cardiomyopathy. A unique entity or a complication of coronary artery disease? Diabetes Care 18: 708–714, 1995.[Abstract]
  5. Cai L. Suppression of nitrative damage by metallothionein in diabetic heart contributes to the prevention of cardiomyopathy. Free Radic Biol Med 41: 851–861, 2006.[CrossRef][Web of Science][Medline]
  6. Chiu HC, Kovacs A, Blanton RM, Han X, Courtois M, Weinheimer CJ, Yamada KA, Brunet S, Xu H, Nerbonne JM, Welch MJ, Fettig NM, Sharp TL, Sambandam N, Olson KM, Ory DS, Schaffer JE. Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy. Circ Res 96: 225–233, 2005.[Abstract/Free Full Text]
  7. Das Evcimen N, King GL. The role of protein kinase C activation and the vascular complications of diabetes. Pharmacol Res 55: 498–510, 2007.[CrossRef][Web of Science][Medline]
  8. Dewald O, Sharma S, Adrogue J, Salazar R, Duerr GD, Crapo JD, Entman ML, Taegtmeyer H. Downregulation of peroxisome proliferator-activated receptor-alpha gene expression in a mouse model of ischemic cardiomyopathy is dependent on reactive oxygen species and prevents lipotoxicity. Circulation 112: 407–415, 2005.[Abstract/Free Full Text]
  9. Dong F, Li Q, Sreejayan N, Nunn JM, Ren J. Metallothionein prevents high-fat diet induced cardiac contractile dysfunction: role of peroxisome proliferator activated receptor gamma coactivator 1alpha and mitochondrial biogenesis. Diabetes 56: 2201–2212, 2007.[CrossRef][Web of Science][Medline]
  10. Escary JL, Choy HA, Reue K, Wang XP, Castellani LW, Glass CK, Lusis AJ, Schotz MC. Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages. J Lipid Res 40: 397–404, 1999.[Abstract/Free Full Text]
  11. Finck BN, Lehman JJ, Leone TC, Welch MJ, Bennett MJ, Kovacs A, Han X, Gross RW, Kozak R, Lopaschuk GD, Kelly DP. The cardiac phenotype induced by PPARalpha overexpression mimics that caused by diabetes mellitus. J Clin Invest 109: 121–130, 2002.[CrossRef][Web of Science][Medline]
  12. Finck BN, Han X, Courtois M, Aimond F, Nerbonne JM, Kovacs A, Gross RW, Kelly DP. A critical role for PPARalpha-mediated lipotoxicity in the pathogenesis of diabetic cardiomyopathy: modulation by dietary fat content. Proc Natl Acad Sci USA 100: 1226–1231, 2003.[Abstract/Free Full Text]
  13. Haemmerle G, Zimmermann R, Hayn M, Theussl C, Waeg G, Wagner E, Sattler W, Magin TM, Wagner EF, Zechner R. Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis. J Biol Chem 277: 4806–4815, 2002.[Abstract/Free Full Text]
  14. Haemmerle G, Lass A, Zimmermann R, Gorkiewicz G, Meyer C, Rozman J, Heldmaier G, Maier R, Theussl C, Eder S, Kratky D, Wagner EF, Klingenspor M, Hoefler G, Zechner R. Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase. Science 312: 734–737, 2006.[Abstract/Free Full Text]
  15. Hamilton JA, Guo W, Kamp F. Mechanism of cellular uptake of long-chain fatty acids: Do we need cellular proteins? Mol Cell Biochem 239: 17–23, 2002.[CrossRef][Web of Science][Medline]
  16. Holm C, Kirchgessner TG, Svenson KL, Fredrikson G, Nilsson S, Miller CG, Shively JE, Heinzmann C, Sparkes RS, Mohandas T, Lusis AJ, Belfrage P, Schotz MC. Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3. Science 241: 1503–1506, 1988.[Abstract/Free Full Text]
  17. Holm C, Osterlund T, Laurell H, Contreras JA. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Annu Rev Nutr 20: 365–393, 2000.[CrossRef][Web of Science][Medline]
  18. Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W, King GL. Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci USA 89: 11059–11063, 1992.[Abstract/Free Full Text]
  19. Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11: 447–455, 1979.[CrossRef][Web of Science][Medline]
  20. Kanehara H, Suzuki J, Zenimaru Y, Takahashi S, Oida K, Shen WJ, Kraemer FB, Miyamori I. Function of hormone-sensitive lipase in diacylglycerol-protein kinase C pathway. Diabetes Res Clin Pract 65: 209–215, 2004.[CrossRef][Web of Science][Medline]
  21. Kang YJ, Chen Y, Yu A, Voss-McCowan M, Epstein PN. Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Invest 100: 1501–1506, 1997.[Web of Science][Medline]
  22. Kraemer FB, Patel S, Saedi MS, Sztalryd C. Detection of hormone-sensitive lipase in various tissues. I. Expression of an HSL/bacterial fusion protein and generation of anti-HSL antibodies. J Lipid Res 34: 663–671, 1993.[Abstract]
  23. Kraemer FB, Shen WJ. Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis. J Lipid Res 43: 1585–1594, 2002.[Abstract/Free Full Text]
  24. Kuriyama H, Liang G, Engelking LJ, Horton JD, Goldstein JL, Brown MS. Compensatory increase in fatty acid synthesis in adipose tissue of mice with conditional deficiency of SCAP in liver. Cell Metab 1: 41–51, 2005.[CrossRef][Web of Science][Medline]
  25. Lucas S, Tavernier G, Tiraby C, Mairal A, Langin D. Expression of human hormone-sensitive lipase in white adipose tissue of transgenic mice increases lipase activity but does not enhance in vitro lipolysis. J Lipid Res 44: 154–163, 2003.[Abstract/Free Full Text]
  26. Maxwell MA, Cleasby ME, Harding A, Stark A, Cooney GJ, Muscat GE. Nur77 regulates lipolysis in skeletal muscle cells. Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway. J Biol Chem 280: 12573–12584, 2005.[Abstract/Free Full Text]
  27. Murphy E, Steenbergen C. Cardioprotection in females: a role for nitric oxide and altered gene expression. Heart Fail Rev 12: 293–300, 2007.[CrossRef][Web of Science][Medline]
  28. Nakai A, Yamaguchi O, Takeda T, Higuchi Y, Hikoso S, Taniike M, Omiya S, Mizote I, Matsumura Y, Asahi M, Nishida K, Hori M, Mizushima N, Otsu K. The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress. Nat Med 13: 619–624, 2007.[CrossRef][Web of Science][Medline]
  29. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95: 351–358, 1979.[CrossRef][Web of Science][Medline]
  30. Park SY, Kim HJ, Wang S, Higashimori T, Dong J, Kim YJ, Cline G, Li H, Prentki M, Shulman GI, Mitchell GA, Kim JK. Hormone-sensitive lipase knockout mice have increased hepatic insulin sensitivity and are protected from short-term diet-induced insulin resistance in skeletal muscle and heart. Am J Physiol Endocrinol Metab 289: E30–E39, 2005.[Abstract/Free Full Text]
  31. Park TS, Yamashita H, Blaner WS, Goldberg IJ. Lipids in the heart: a source of fuel and a source of toxins. Curr Opin Lipidol 18: 277–282, 2007.[Web of Science][Medline]
  32. Poornima IG, Parikh P, Shannon RP. Diabetic cardiomyopathy: the search for a unifying hypothesis. Circ Res 98: 596–605, 2006.[Abstract/Free Full Text]
  33. Preiss J, Loomis CR, Bishop WR, Stein R, Niedel JE, Bell RM. Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem 261: 8597–8600, 1986.[Abstract/Free Full Text]
  34. Pruimboom-Brees I, Haghpassand M, Royer L, Brees D, Aldinger C, Reagan W, Singh J, Kerlin R, Kane C, Bagley S, Hayward C, Loy J, O'Brien P, Francone OL. A critical role for peroxisomal proliferator-activated receptor-alpha nuclear receptors in the development of cardiomyocyte degeneration and necrosis. Am J Pathol 169: 750–760, 2006.[Abstract/Free Full Text]
  35. Regan TJ, Lyons MM, Ahmed SS, Levinson GE, Oldewurtel HA, Ahmad MR, Haider B. Evidence for cardiomyopathy in familial diabetes mellitus. J Clin Invest 60: 884–899, 1977.[Medline]
  36. Rosenkranz S. TGF-beta1 and angiotensin networking in cardiac remodeling. Cardiovasc Res 63: 423–432, 2004.[Abstract/Free Full Text]
  37. Rubler S, Dlugash J, Yuceoglu YZ, Kumral T, Branwood AW, Grishman A. New type of cardiomyopathy associated with diabetic glomerulosclerosis. Am J Cardiol 30: 595–602, 1972.[CrossRef][Web of Science][Medline]
  38. Son NH, Park TS, Yamashita H, Yokoyama M, Huggins LA, Okajima K, Homma S, Szabolcs MJ, Huang LS, Goldberg IJ. Cardiomyocyte expression of PPARgamma leads to cardiac dysfunction in mice. J Clin Invest 117: 2791–2801, 2007.[CrossRef][Web of Science][Medline]
  39. Subramaniam A, Gulick J, Neumann J, Knotts S, Robbins J. Transgenic analysis of the thyroid-responsive elements in the alpha-cardiac myosin heavy chain gene promoter. J Biol Chem 268: 4331–4336, 1993.[Abstract/Free Full Text]
  40. Suzuki J, Kanehara H, Takahashi S, Oida K, Osuga J, Ishibashi S, Shen WJ, Kraemer FB, Miyamori I. Paradoxical regulation of cardiac lipases and lipid accumulation in hormone-sensitive lipase knockout mice (Abstract). Diabetes 53, Suppl 2: A338, 2004.
  41. Suzuki J, Shen WJ, Nelson BD, Patel S, Veerkamp JH, Selwood SP, Murphy GM Jr, Reaven E, Kraemer FB. Absence of cardiac lipid accumulation in transgenic mice with heart-specific HSL overexpression. Am J Physiol Endocrinol Metab 281: E857–E866, 2001.[Abstract/Free Full Text]
  42. Suzuki J, Shen WJ, Nelson BD, Selwood SP, Murphy GM Jr, Kanehara H, Takahashi S, Oida K, Miyamori I, Kraemer FB. Cardiac gene expression profile and lipid accumulation in response to starvation. Am J Physiol Endocrinol Metab 283: E94–E102, 2002.[Abstract/Free Full Text]
  43. Sztalryd C, Kraemer FB. Regulation of hormone-sensitive lipase in streptozotocin-induced diabetic rats. Metabolism 44: 1391–1396, 1995.[CrossRef][Web of Science][Medline]
  44. Unger RH, Orci L. Diseases of liporegulation: new perspective on obesity and related disorders. FASEB J 15: 312–321, 2001.[Abstract/Free Full Text]
  45. Uno K, Katagiri H, Yamada T, Ishigaki Y, Ogihara T, Imai J, Hasegawa Y, Gao J, Kaneko K, Iwasaki H, Ishihara H, Sasano H, Inukai K, Mizuguchi H, Asano T, Shiota M, Nakazato M, Oka Y. Neuronal pathway from the liver modulates energy expenditure and systemic insulin sensitivity. Science 312: 1656–1659, 2006.[Abstract/Free Full Text]
  46. Valmaseda A, Carmona MC, Barbera MJ, Vinas O, Mampel T, Iglesias R, Villarroya F, Giralt M. Opposite regulation of PPAR-alpha and -gamma gene expression by both their ligands and retinoic acid in brown adipocytes. Mol Cell Endocrinol 154: 101–109, 1999.[CrossRef][Web of Science][Medline]
  47. Westermann D, Rutschow S, Jager S, Linderer A, Anker S, Riad A, Unger T, Schultheiss HP, Pauschinger M, Tschope C. Contributions of inflammation and cardiac matrix metalloproteinase activity to cardiac failure in diabetic cardiomyopathy: the role of angiotensin type 1 receptor antagonism. Diabetes 56: 641–646, 2007.[Abstract/Free Full Text]
  48. Winzell MS, Svensson H, Enerback S, Ravnskjaer K, Mandrup S, Esser V, Arner P, Alves-Guerra MC, Miroux B, Sundler F, Ahren B, Holm C. Pancreatic beta-cell lipotoxicity induced by overexpression of hormone-sensitive lipase. Diabetes 52: 2057–2065, 2003.[Abstract/Free Full Text]
  49. Wold LE, Ceylan-Isik AF, Fang CX, Yang X, Li SY, Sreejayan N, Privratsky JR, Ren J. Metallothionein alleviates cardiac dysfunction in streptozotocin-induced diabetes: role of Ca2+ cycling proteins, NADPH oxidase, poly(ADP-Ribose) polymerase and myosin heavy chain isozyme. Free Radic Biol Med 40: 1419–1429, 2006.[CrossRef][Web of Science][Medline]
  50. Yagyu H, Chen G, Yokoyama M, Hirata K, Augustus A, Kako Y, Seo T, Hu Y, Lutz EP, Merkel M, Bensadoun A, Homma S, Goldberg IJ. Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy. J Clin Invest 111: 419–426, 2003.[CrossRef][Web of Science][Medline]
  51. Zhou YT, Grayburn P, Karim A, Shimabukuro M, Higa M, Baetens D, Orci L, Unger RH. Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci USA 97: 1784–1789, 2000.[Abstract/Free Full Text]
  52. Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A, Zechner R. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306: 1383–1386, 2004.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
J. Suzuki, M. Ueno, M. Uno, Y. Hirose, Y. Zenimaru, S. Takahashi, J.-i. Osuga, S. Ishibashi, M. Takahashi, M. Hirose, et al.
Effects of hormone-sensitive lipase disruption on cardiac energy metabolism in response to fasting and refeeding
Am J Physiol Endocrinol Metab, November 1, 2009; 297(5): E1115 - E1124.
[Abstract] [Full Text] [PDF]

Home page
 DMMHome page
H. Bugger and E. D. Abel
Rodent models of diabetic cardiomyopathy
Dis. Model. Mech., September 1, 2009; 2(9-10): 454 - 466.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/6/E1109    most recent
00016.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ueno, M.
Right arrow Articles by Miyamori, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ueno, M.
Right arrow Articles by Miyamori, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.